Granulosa Cells Of Graafian Follicles Research Articles

The concentration-dependent effect of progesterone on follicle growth in the mouse ovary.

Follicle growth in the mammalian ovary is coordinately controlled by multiple factors to sustain periodic ovulation. In this study, we investigated the role of progesterone on follicle growth in the mouse ovary. As the concentration of progesterone changes during the estrus cycle, we cultured the sliced mouse ovary in a medium containing 10 ng/ml, 100 ng/ml, and 1 μg/ml progesterone. Progesterone promoted the growth of primordial to primary follicles at 100 ng/ml, while it suppressed the growth of secondary follicles at 1 μg/ml. Follicles at other developmental stages in the cultured ovary were unaffected with different concentrations of progesterone. The number of ovulated oocytes increased in the medium containing 100 ng/ml progesterone but decreased in the presence of 1 μg/ml progesterone. Follicles expressed two types of progesterone receptors, progesterone receptor (PGR) and PGR membrane component 1 (PGRMC1). While PGR shows transient expression on granulosa cells of Graafian follicles, PGRMC1 expresses in granulosa cells of developing follicles. These results suggest that progesterone controls the growth of developing follicles through PGRMC1. Our study shows that the effect of progesterone on ovulation and follicle growth in mouse ovary is dependent on the concentration of progesterone and the follicle stage.

Comparison of transforming growth factor β1 concentrations in the ovaries of rats stimulated by human menopausal gonadotropin or recombinant follicle-stimulating hormone

To compare the effects of human menopausal gonadotropins (hMG) and recombinant follicle stimulating hormone (rFSH) on transforming growth factor (TGF) β1 concentration in the rat ovary. Twenty-one fertile Wistar-Albino rats were divided into 3 groups of 7. Groups 1, 2 and 3 were injected with saline, hMG or rFSH, respectively, over 5 days, after which they underwent ovariectomy. Hematoxylin and eosin (H&E) staining was used for histological examination. TGF β1 staining levels in ovarian stroma, vessel walls, granulosa cells of Graafian follicles and corpus luteum cells were investigated immunohistochemically. On histological examination, the number of smaller antral follicles was higher in the control group, while there were more and larger antral follicles in the hyperstimulated groups. There were statistically significant differences in staining in vessel walls and granulosa cells between the control and stimulated groups. Both stimulation protocols caused an increased TGF β1 concentration in vessel walls, while there was weak staining in granulosa cells in the treatment groups compared to the control group (p<0.05). There were no significant differences in staining scores between the two treatment groups (p>0.05). The effects of two different gonadotropin preparations on TGF β1 concentrations in different localizations in the rat ovaries are comparable. It may be postulated that the luteinizing hormone (LH) content of hMG contributes little or nothing to the TGF β1 mediated angiogenesis.

Spatio-temporal expression patterns of steroidogenic acute regulatory protein (StAR) during follicular development in the rat ovary.

The steroidogenic acute regulatory protein (StAR) is a vital mitochondrial protein that is indispensable for the synthesis of steroid hormones in the steroidogenic cells of the adrenal cortex and the gonads. Recent studies have shown that StAR enhances the conversion of the substrate for all steroid hormones, cholesterol, into pregnenolone, probably by facilitating cholesterol entry into the inner compartment of the mitochondria where the steroidogenic cytochrome P450scc complex resides. To study the potential of StAR to affect ovarian steroidogenesis during follicular development, we examined the time-dependent expression of StAR protein and messenger RNA in PMSG/human CG (hCG)-treated immature rats. Western blot analyses and immunohistochemical and RT-PCR methodologies have revealed a biphasic expression of StAR in the ovaries responding to hormones. The first peak of StAR expression was generated by PMSG administration and lasted for 24 h. Furthermore, it was restricted to the entire network of the ovarian secondary interstitial tissue, as well as to a fewer scattered theca-interna cells. The second burst of StAR expression was observed in response to the LH surge, as simulated by hCG. This time, StAR was expressed in the entire theca-interna and interstitial tissue, as well as in those granulosa cells that were confined to periovulatory follicles. Immunoelectron microscopy studies revealed the over 90% of StAR antigenic sites are localized in the inner compartments of the mitochondrion, suggesting a rapid removal of StAR precursor from the mitochondrial surface, where it is believed to exert its activity. Altogether, our observations portray dynamic acute alterations of StAR expression during the process of follicular maturation in this animal model. Furthermore, if StAR indeed determines steroidogenic capacities in the ovary, our findings imply that, in immature rats undergoing hormonally induced first ovulation: 1) the early phases of follicular development are supported by androgen production originating from nonfollicular cells; 2) estrogen production in the granulosa cells of Graafian follicles is nourished by a submaximal androgenic output in the theca-interstitial compartments of the ovary.

Expression of human group II phospholipase A2 in transgenic mice.

Group II phospholipase A2 (PLA2) has been proposed to play an important role in inflammation and defense against bacterial infection. We investigated tissues of transgenic mice expressing the human group II PLA2 gene by immunohistochemistry using rabbit anti-human group II PLA2 antibodies, and by in situ hybridization by probing with human group II PLA2 mRNA anti-sense (test) and sense (control) riboprobes. By immunohistochemistry, human group II PLA2 was found in various mouse tissues and cell types including hepatocytes, proximal tubule cells of the kidney, epithelial cells of the renal pelvis, urinary bladder and ureter, granulosa cells of Graafian follicles, aortic intima and media, cartilage, epiphyseal bone, bronchial epithelial cells, and connective tissue cells in the dermis. By in situ hybridization, group II PLA2 mRNA was localized in hepatocytes, epidermal cells, dermal cells, connective tissue fibroblasts, epithelial and smooth muscle cells of the urinary bladder, and cells of Bowman's capsule. These results show that human group II PLA2 is expressed in large amounts in hepatocytes and many extrahepatic tissues of the transgenic mice. These animals provide a useful new tool for studies on the metabolism, in vivo effects, and physiological and pathological roles of phospholipase A2.

Aetiology of intersexuality in female (XX) pigs, with novel molecular interpretations.

This review considers the problem of ovotestis formation in animals of 38,XX chromosome complement. After a clinical description, attention focuses on the condition of the gonads and genital tract. A complete spectrum of gonadal types has been found, ranging from a single ovotestis almost invariably on the right-hand side to both gonads appearing as testicular-like structures, sometimes with a distinct tunica albuginea. The ovotestis or testis-like structure may have descended to an inguinal or scrotal location. Although interstitial cells of Leydig and seminiferous tubules were always abundant in testicular tissue, germ cells were never present. The lumen of the seminiferous tubules was packed with pale-staining, Sertoli-like cells. A bicornuate uterus was characteristic but suppression of the proximal portion of the Müllerian duct always adjoined an ovotestis; a corresponding development of the Wolffian duct featured as a convoluted epididymis. Inhibition of the Fallopian tube was attributed to a local influence of AMH from the Sertoli cells, as was the failure of small Graafian follicles within an ovotestis to respond to injected gonadotrophins. As to the aetiology of an ovotestis, defective colonisation of the genital ridges by primordial germ cells is considered, as is evidence for incorporation of adrenal cells into the embryonic gonad. Molecular probing has failed to reveal the classical sex-determining gene, Sry, and other Y-related DNA sequences such as Zfy and DYZI in almost all the intersex animals examined. Currently favoured as an explanation for ovotestis formation is a mutation in the inhibin gene within granulosa cells of Graafian follicles. Such a mutation would prompt secretion of the closely comparable glycoprotein molecule AMH in these genetic females, with a resultant progressive virilisation of gonadal tissue. The proposed mutation may be carried as an autosomal recessive gene by certain boars. Varying amounts of AMH secretion or differing timescales for the transition from inhibin to AMH could in part explain differing degrees of ovotestis formation. Despite this proposition, interactions between genes that prescribe functional testicular tissue, enhanced rates of gonadal development, and left-right asymmetries between the paired gonads now require systematic study.

Cell-specific expression of aromatase and LH receptor mRNAs in rat ovary.

Current understanding of the endocrine and paracrine regulation of follicular oestrogen synthesis predicts that aromatase cytochrome P450 (P450arom) mRNA is inducible by FSH in granulosa cells. LH receptor mRNA is constitutively expressed in thecal/interstitial cells, and is also thought to be induced in granulosa cells in response to joint stimulation by FSH and oestrogen. This study provides direct evidence that FSH induces the ovarian P450arom gene selectively, perhaps exclusively, in the granulosa cells of Graafian follicles. FSH-induction of LH receptor mRNA occurs simultaneously but is independent of oestrogen synthesis per se.

Cellular pattern of insulin-like growth factor-I (IGF-I) and IGF-I receptor gene expression in the developing and mature ovarian follicle.

We have employed in situ hybridization histochemistry to map the cellular pattern of insulin-like growth factor-I (IGF-I) and IGF-I receptor gene expression in developing rat ovaries from the time of birth through adulthood, and in response to hypophysectomy and gonadotropin replacement. From the early postnatal period, both IGF-I and IGF-I receptor messenger RNAs (mRNAs) were highly abundant and evenly distributed in granulosa cells of small, growing follicles. In large follicles, however, IGF-I gene expression was heterogeneous. IGF-I mRNA was most abundant in granulosa cells lining the antrum and surrounding the oocyte, but was low or undetectable in mural granulosa cells of Graafian follicles, and was also undetectable in luteinized granulosa cells of corpora lutea. IGF-I receptor mRNA was evenly distributed in developing and mature follicles and was highly abundant in the luteinized granulosa cells of corpora lutea. IGF-I receptor but not IGF-I mRNA was detected in growing oocytes. Hypophysectomy resulted in a decrease and treatment with PMSG resulted in an increase in follicular IGF-I receptor mRNA levels, whereas there was no change in IGF-I mRNA levels in the same protocol. In summary, high levels of both IGF-I and IGF-I receptor gene expression occur in the granulosa cells of actively growing follicles, suggesting that granulosa cell IGF-I may have a role in follicular or oocyte growth. IGF-I gene expression is lost concomitant with follicular enlargement and granulosa cell differentiation, whereas IGF-I receptor gene expression continues at high levels in luteinized granulosa cells, suggesting that IGF effects on differentiated granulosa cell function are due to circulating, not local, hormone. Finally, granulosa cell IGF-I receptor gene expression appears to be regulated by the gonadotropin present in pregnant mare serum.

Immunocytochemical Localization and Enzymatic Distribution of Rat Ovarian Aromatase1

A rabbit antiserum directed against purified human placental aromatase was used for immunohistochemical localization of the enzyme in rat ovaries. Immunostaining was conducted on tissue from animals at various ages and in different reproductive states: immature; immature, eCG-treated; immature pseudopregnant; adult cycling; and adult pregnant. Various labeling protocols were employed (e.g. horseradish peroxidase-conjugated secondary antibody, peroxidase-antiperoxidase, and avidin-biotin-peroxidase on fresh frozen and Bouin's fixed paraffin-embedded sections), but the avidin-biotin-peroxidase method on paraffin sections proved to be superior to the others. In immature rats, most of the immunostaining, which was quite weak, was limited to the stroma. After stimulation with eCG, some of the granulosa cells of antral follicles exhibited immunostaining; in pseudopregnant rats, most staining occurred in the luteal cells. In mature animals, the corpora lutea of pregnant and cycling rats demonstrated the greatest degree of immunostaining. No significant immunoreactivity was detected in pre-antral follicles, but in early antral follicles and preovulatory follicles, both theca and granulosa cells exhibited immunostaining. Aromatase enzymatic activity was also determined on ovarian microsomal fractions of eCG-treated immature animals, pregnant animals at term, and cycling animals. Furthermore, enzyme activity and estradiol concentrations were examined after ovaries from proestrous rats were dissected into follicular, luteal, and residual components. Activity was found in all regions and correlated with immunostaining and estrogen production. These results argue against a model in which all the immunoreactive/enzymatically active protein is localized in granulosa cells of Graafian follicles and suggest that corpora lutea may be involved in estrogen synthesis during the rat estrous cycle as well as during pregnancy.

LHRH stimulates plasminogen activator and inhibits steroid production by granulosa cells of adult rat Graafian follicles

We studied the effects of LHRH and its analogs on plasminogen activator production and progesterone and estradiol secretion by granulosa cells isolated from adult rat Graafian follicles. LHRH and its agonist ([des-Gly 10, d-Trp 6, Pro 9-NHEt]LHRH) stimulated small but significant increases in plasminogen activator production. This stimulatory action of LHRH was blocked by the addition of the specific antagonist ([ d -p Glu 1 , d-Phe 2, d-Trp 3.6]LHRH). In contrast, LHRH treatment had no significant effect on basal steroid production by granulosa cells of Graafian follicles cultured for 2–6 days. However, LHRH decreased the FSH-stimulated steroidogenesis. This inhibitory effect of LHRH on steroidogenesis was abolished by concomitant addition of the LHRH antagonist. The results show that LHRH has a stimulatory effect on plasminogen activator production and a suppressive action on steroidogenesis in granulosa cells isolated from Graafian follicles adult rats.

Identification of subpopulations of rat granulosa cells: sedimentation properties and hormonal responsiveness.

Ovarian granulosa cells from small follicles have generally been considered to comprise a homogeneous cell population; however, stratified arrangements of hormone receptors have been found in antral and mural granulosa cells of Graafian follicles. Using cultured granulosa cells derived from immature, hypophysectomized, estrogen-treated rats, we have previously shown that vasoactive intestinal peptide (VIP) as well as FSH stimulates steroid production by these cells. Dose-response analysis indicated that the actions of these hormones were additive, suggesting the presence of subpopulations of granulosa cells. Using a continuous (0-30%) Metrizamide density gradient, we have identified three populations of granulosa cells with different sedimentation properties. After centrifugation for 15 min at 1500 X g, cells sedimented at Metrizamide concentrations of 13%, 18%, and 20% (peaks A, B, and C, respectively). The subpopulation with lowest density (peak A) comprised 5% of the total cells, whereas the remaining cells were distributed approximately equally in the other two peaks. The profiles of estrogen and progesterone production by these cells in response to FSH and VIP indicated that FSH preferentially stimulated steroid production in cells with the highest density (peak C), whereas VIP mainly induced steroidogenic responses in cells of intermediate density (peak B). In contrast, cells with the lowest density (peak A) were unresponsive to either hormone. Treatment with forskolin, a universal adenylate cyclase activator, induced steroid production in both subpopulations B and C. Further studies demonstrated that LH/human CG receptors were induced by FSH and forskolin in cells from peak C, whereas VIP treatment did not induce LH/human CG receptors in cells from peak B. In unfractionated cultured cells, GnRH potently antagonized FSH- but not VIP-induced steroidogenesis. Upon density-gradient fractionation, the profile of GnRH receptor content correlated well with GnRH effects since FSH-responsive cells (peak C) contained the majority of GnRH receptors. The present results demonstrate that granulosa cells from immature follicles are heterogeneous and consist of two major subpopulations of cells with differential responsiveness to FSH and VIP. These findings provide the basis for further morphological and biochemical analysis of subpopulations of granulosa cells during follicular development.

Divergent effects of prolactin on estrogen and progesterone production by granulosa cells of rat Graafian follicles.

The effect of PRL on ovarian steroidogenesis was studied in cultured granulosa cells isolated from follicles of mature cycling rats on the morning of proestrus. Ovine PRL 10-1000 ng/ml) inhibited estradiol production but stimulated progesterone biosynthesis in a dose-dependent manner. The effect of PRL was most prominent after 4 days of culture: 1000 ng/ml PRL suppressed estradiol production by 80% but increased progesterone synthesis by 290%, whereas the lower dose of 10 ng/ml inhibited estrogen secretion by 20% without altering progesterone synthesis. The divergent effect of PRL was not shown to be species specific, since ovine, rat and human PRL had similar effects. Using increasing concentrations of androstenedione (the aromatase substrate), estrogen secretion remained suppressed and progesterone production was stimulated by PRL. FSH stimulated both estrogen and progesterone production. The FSH-induced increased in estrogen production was inhibited by concomitant treatment with PRL. In contrast, PRL and FSH had an additive action in stimulating progesterone production. Although LH alone had no effect on steroidogenesis, concomitant treatment with LH and PRL resulted in a stimulation of progesterone production that was additive. This study demonstrates that PRL acts directly on granulosa cells of Graafian follicles of adult cycling rats to stimulate the secretion of progesterone and to suppress estradiol production.